Mycobacterium tuberculosis (Mtb) is the leading infectious disease-related cause of death among people living with HIV worldwide. Shorter tuberculosis (TB) treatment regimens are needed to achieve global TB elimination. The protracted nature of the current 6-month TB treatment course reflects the unique ability of a subpopulation of ?persister? bacteria to remain in a growth-limited, antibiotic-tolerant state through various adaptive strategies, including induction of the stringent response. The key stringent response enzyme RelMtb is essential for long-term Mtb survival under physiologically relevant stresses in vitro and in animal lungs. Recently, we have generated a therapeutic relMtb DNA vaccine, which induces RelMtb-specific cellular immunity, and significantly augments the activity of the first-line drug isoniazid against active TB in mice. We also have developed a novel vaccination strategy involving fusion of the antigen of interest with the immature dendritic cell (iDC)-targeting chemokine MIP3?, which significantly enhances antigen-specific T-cell responses. In the current proposal, we will determine if this iDC-targeting strategy, as well as a promising new adjuvant approach involving the use of cyclic dinucleotides to activate the stimulator of interferon genes (STING) pathway, enhance the immunogenicity of our relMtb DNA vaccine. The ideal vaccine platform will be used to test the novel hypothesis that enhanced cellular immunity against RelMtb potentiates the activity of the first- line anti-TB regimen and accelerates cure in the standard murine model of TB. Since HIV infection is associated with disturbed T-cell homeostasis, including depletion of CD4+ T cells and persistent expansion of CD8+ T cells, we will characterize the contribution of each of these cell types to the therapeutic efficacy of the relMtb DNA vaccine in mice. In order to transition our findings to the clinical setting, we will next test the immunogenicity of this vaccination strategy in rhesus macaques, which develop immune response patterns most analogous to those of humans. Finally, leveraging archived clinical samples available through the RePORT South Africa longitudinal cohort of HIV-infected and uninfected patients with pulmonary TB receiving first-line anti-tubercular treatment, we will measure RelMtb-specific T-cell responses during TB therapy. This proposal represents a unique collaboration between Investigators with significant expertise in microbiology, molecular biology, immunology, DNA vaccines, and animal models. Our findings are expected to have far- reaching implications for the development of novel adjunctive therapies for shortening the duration of treatment for drug-susceptible and drug-resistant TB, as well as novel diagnostic tools for confirming the adequacy of TB treatment in HIV-infected and uninfected individuals.